Figure 7. Outline of tbe rauiioe bone marrow (ranjplantatioo/gene transfer protocol. Bone mar- 
row (BM) cells are isolated from the femur, exposed to the vector by co-cultivation with vector pro- 
ducer cells, and then introduced into lethally irradiated recipient mice. CFU-S are progenitor cells 
whose lineage n assayable by colonies (or foci) formed in the spleen. The number of spleen foci 
are proportional to the total number of stem cells injected. 
ihc template, they wiU be absent from 
both LTRs in the target cells. 
The structure of a prototype SIN vec- 
tor is shown at the top of Figure 6. The 
selectable gene is expressed from an in- 
ternal promoter (cloned into the vector 
along with the gene) rather than from 
the viral LTR as with the VIP vectors 
discussed in the previous section. Since, 
upon infection of the target cells, the 
promoter activity in the viral LTR is in- 
activated, the expression of the selec- 
table gene in the target cells is controlled 
by its own internal promoter. SV-N and 
MT-N are two examples of SIN vectors 
shown in the lower portion of Fig. 6; the 
early S V40 (SV) or mouse metallothio- 
nein I (MT) promoters are used respec- 
tively to drive the expression of the 
selectable gene coding for neomycin 
resistance (Neo*). The gene of interest 
can be inserted into SIN vectors either 
in front of or behind the selectable 
marker, utilizing the presence of unique 
restriction sites (BamHI and Xhol, 
respectively. Fig. 6). 
Although it has been demonstrated 
that SIN vectors indeed self-activate in 
the target cell (29), the titer of virus 
generated from this type of vector are 
disappointingly low (~10* cfu/ml), 
probably not sufficient for use in 
human therapy. It is hoped that ap- 
propriate modifications, together with 
advances in the understanding of the 
retroviral genome, will increase the per- 
formance of SIN vectors. 
EXPRESSION 
Retroviral vectors are much more ef- 
ficient for the insertion of DNA sequen- 
ces than other presently available means 
of gene transfer. They are valuable not 
only for cells growing attached to a 
substratum, but also for cells grown in 
suspension culture. This latter quality 
has given geneticists access to a great 
many types of cells previously refrac- 
tory to genetic engineering. 
If hematopoietic stem cells could be 
infected with a vector, the potential ex- 
ists for new genetic material to become 
a permanent pan of the blood tissue’s 
genetic complement. Stem cells are 
most abundant within the bone mar- 
row. Yet even there they are present 
only as a tiny fraction (less than one cell 
in a thousand) of the total nucleated 
cells. It is only by means of retroviral 
vectors, with their combined character- 
istics of very high efficiency and ap- 
plicability to cells in suspension, that 
genetic manipulation of the totipotent 
stem cells of the hematopoietic system 
has become practical. 
Several laboratories have published 
protocols by which the hematopoietic 
cells of mice have had new genes in- 
troduced with retroviral vectors ex vivo, 
with subsequent reintroduction of the 
treated cells into the bone marrow in 
vivo. The basic outline of such a bone 
marrow transplantation/gene transfer 
protocol is very simple (Fig. 7). Marrow 
cells are obtained from a donor and in- 
cubated with a monolayer of vector- 
generating producer cells (27). Since 
marrow cells, unlike the producer cells, 
do not attach to the culture dish, they 
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Recombinant DNA Research, Volume 12 
